Beta-Thalassemia

[Cooley's Anemia, Mediterranean Anemia. Includes: Thalassemia Major (Beta-Thalassemia Major), Thalassemia Intermedia, Thalassemia Minor (Beta-Thalassemia Minor, Heterozygous Beta-Thalassemia)]

Clinical Diagnosis

Thalassemia major is suspected in an infant or child younger than age two years with severe microcytic anemia and hepatosplenomegaly. Untreated, affected children usually manifest failure to thrive and expansion of the bone marrow to compensate for ineffective erythropoiesis.

Thalassemia intermedia is suspected in individuals who present at a later age with a milder anemia that only rarely requires treatment with blood transfusion.

Testing

Molecular Genetic Testing

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Gene. Mutations in HBB are associated with β-thalassemia (see Differential Diagnosis).

Clinical testing

• Targeted mutation analysis. The β-thalassemias can be caused by more than 200 different HBB gene mutations [Huisman et al 1997, globin.cse.psu.edu]; however, the prevalent molecular defects are limited in each at-risk population (see Table 4). This phenomenon has greatly facilitated molecular genetic testing.

  • Commonly occurring mutations of the HBB gene are detected by a number of PCR-based procedures. The most commonly used methods are reverse dot blot analysis or primer-specific amplification, with a set of probes or primers complementary to the most common mutations in the population from which the affected individual originated [Old et al 2005].

  • Other methods based on real-time PCR or microarray technology because of their reproducibility, rapidity, and easy handling are potentially suitable for the routine clinical laboratory [Vrettou et al 2003, Ye et al 2007].

• Mutation scanning/sequence analysis. If targeted mutation analysis fails to detect the mutation, mutation scanning or sequence analysis can be used to detect mutations in the HBB coding region (mutations in the non-coding region would not be detected by this analysis). Sensitivity of both mutation scanning and sequence analysis is 99%.

Note: Deletions of variable extent of the β gene or of the HBB cluster that result in β-thalassemia or in the complex β-thalassemias called γδβ-thalassemia and δβ-thalassemia are rare causes of β-thalassemia and testing that detects deletions is available clinically.

Table 3 summarizes molecular genetic testing for this disorder.

Table 3. Molecular Genetic Testing Used in Beta-Thalassemia

Test Method	Mutations Detected	Mutation Detection Frequency 1	Test Availability
Targeted mutation analysis	HBB mutation panels vary by laboratory	Variable depending on mutations included in panel and individual's ethnicity	Clinical
Mutation scanning/ sequence analysis	Sequence variants in HBB coding region	99%

1. Proportion of affected individuals with a mutation(s) as classified by test method

Interpretation of test results. For issues to consider in interpretation of sequence analysis results, click here.

Testing Strategy

Confirmation of the diagnosis of beta-thalassemia in a proband requires identification of the disease-causing point mutations in HBB. The appropriate order for molecular genetic testing:

• 1

  Targeted mutation analysis

• 2

  Mutation scanning or sequence analysis

Prognostication. Distinguishing thalassemia major from thalassemia intermedia at the molecular level for the purpose of prognostication requires defining of the HMM mutations and evaluating for coinheritance of those genetic determinants able to sustain a continuous production of gamma globin chains (HbF) in adult life or able to reduce the alpha/non-alpha globin chain imbalance, such as alpha-thalassemia (see Genotype-Phenotype Correlations).

Carrier testing for at-risk relatives requires prior identification of the disease-causing mutations in the family.

Note: Carriers are heterozygotes for an autosomal recessive disorder and are not at risk of developing the disorder.

Prenatal diagnosis and preimplantation genetic diagnosis (PGD) for at-risk pregnancies require prior identification of the disease-causing mutation in the family.

Genetically Related (Allelic) Disorders

Other phenotypes associated with mutations in the HBB gene are sickle cell disease caused by A>T substitution at codon 6 (p.Glu6Val) (c.17A>T, Genbank NM_000518.4) and other nucleotide substitutions responsible for hemoglobin variants (globin.cse.psu.edu).

Natural History

The phenotypes of the homozygous β-thalassemias include thalassemia major and thalassemia intermedia. The clinical severity of the β-thalassemia syndromes depends on the extent of globin alpha chain/non-globin alpha chain (i.e., β⁺ γ) imbalance. The non-assembled globin alpha chains that result from unbalanced globin alpha chain/non-globin alpha chain synthesis precipitate in the form of inclusions. These globin alpha chain inclusions damage the erythroid precursors in the bone marrow and in the spleen, causing ineffective erythropoiesis. Individuals with thalassemia major usually come to medical attention within the first two years of life; they subsequently require regular red blood cell transfusions to survive. Those who present later and rarely require transfusion are said to have thalassemia intermedia.

β-thalassemia major. Clinical presentation of thalassemia major occurs between ages six and 24 months. Affected infants fail to thrive and become progressively pale. Feeding problems, diarrhea, irritability, recurrent bouts of fever, and progressive enlargement of the abdomen caused by splenomegaly may occur. If the diagnosis of thalassemia major is established at this stage and if a regular transfusion program that maintains a minimum Hb concentration of 95 to 105 g/L is initiated, growth and development are normal until age ten to 11 years.

After age ten to 11 years, affected individuals are at risk of developing severe complications related to iron overload, depending on their compliance with chelation therapy (see Management). Complications of iron overload in children include growth retardation and failure of sexual maturation as well as those complications observed in adults with HFE-associated hereditary hemochromatosis: involvement of the heart (dilated myocardiopathy and pericarditis), liver (fibrosis and cirrhosis), and endocrine glands (resulting in diabetes mellitus and insufficiency of the parathyroid, thyroid, pituitary, and, less commonly, adrenal glands). In individuals who have been regularly transfused, iron overload results mainly from transfusions. Other complications are hypersplenism, chronic hepatitis (resulting from infection with the viruses that cause hepatitis B and/or hepatitis C), cirrhosis (from iron overload and chronic hepatitis), HIV infection, venous thrombosis, and osteoporosis. The risk for hepatocellular carcinoma is increased secondary to liver viral infection, iron overload, and longer survival [Borgna-Pignatti et al 2004].

The survival of individuals who have been well transfused and treated with appropriate chelation extends beyond age 30 years. Myocardial disease caused by transfusional siderosis is the most important life-limiting complication of iron overload in beta-thalassemia. In fact, cardiac complications are reported to cause 71% of the deaths in individuals withβ-thalassemia major [ Borgna-Pignatti et al 2004].

The classic clinical picture of thalassemia major is presently only seen in some developing countries, in which the resources for carrying out long-term transfusion programs are not available. The most relevant features of untreated or poorly transfused individuals are growth retardation, pallor, jaundice, brown pigmentation of the skin, poor musculature, genu valgum, hepatosplenomegaly, leg ulcers, development of masses from extramedullary hematopoiesis, and skeletal changes that result from expansion of the bone marrow. These skeletal changes include deformities of the long bones of the legs and typical craniofacial changes (bossing of the skull, prominent malar eminence, depression of the bridge of the nose, tendency to a mongoloid slant of the eye, and hypertrophy of the maxillae, which tends to expose the upper teeth). Individuals who have not been regularly transfused usually die before the third decade. Individuals who have been poorly transfused are also at risk for complications of iron overload.

β-thalassemia intermedia. Clinical features are pallor, jaundice, cholelithiasis, liver and spleen enlargement, moderate to severe skeletal changes, leg ulcers, extramedullary masses of hyperplastic erythroid marrow, a tendency to develop osteopenia and osteoporosis, and thrombotic complications resulting from iron accumulation and hypercoagulable state resulting from the lipid membrane composition of the abnormal red blood cells [Eldor & Rachmilewitz 2002]. By definition, transfusions are not required or only occasionally required. Iron overload occurs mainly from increased intestinal absorption of iron caused by ineffective erythropoiesis. The associated complications of iron overload present later, but may be as severe as those seen in individuals with thalassemia major who depend on transfusions.

Genotype-Phenotype Correlations

Any inherited or acquired condition that reduces the alpha/non-alpha globin chain imbalance in β-thalassemia results in a lesser degree of globin alpha chain precipitation and leads to a mild β-thalassemia phenotype [Galanello & Cao 1998].

One of the most common and consistent mechanisms is homozygosity or compound heterozygosity for two β⁺-thalassemia mild and silent mutations (see Table 5).

In contrast, compound heterozygosity for a mild/silent β⁺ and a severe mutation produces a variable phenotype, ranging from thalassemia intermedia to thalassemia major. Therefore, the presence of this genotype does not predict a mild phenotype. Hemoglobin E (HbE), which is a thalassemic structural variant, characterized by the presence of an abnormal structure as well as biosynthetic defect, should be included in this group. The nucleotide substitution at codon 26, producing the HbE variant (α₂ β₂26 E>K), activates a potential cryptic RNA splice region, resulting in alternative splicing at this position. The homozygous state for HbE results in a mild hemolytic microcytic anemia. Compound heterozygosity for β-thalassemia and HbE result in a wide range of often severe but sometimes mild or even clinically asymptomatic clinical phenotypes.

The clinical picture resulting from homozygosity for β⁺-thalassemia or homozygosity for βº-thalassemia mutations may be ameliorated by coinheritance of mutations in the gene encoding the globin alpha chain associated with α-thalassemia, which reduces the output of the genes encoding the globin alpha chains and therefore decreases the alpha/non-alpha globin chain imbalance. Because coinheritedα-thalassemia does not always produce a consistent effect, it cannot be used to predict phenotype.

The coinheritance of some genetic determinants able to sustain a continuous production of globin gamma chains (HbF) in adult life may also reduce the extent of alpha/non-alpha globin chain imbalance:

• The β-thalassemia mutation per se increases the globin gamma chain (HbF) output. This occurs in the following two situations:

  • δβº-thalassemia is caused by deletions of variable size in the HBB gene cluster.

  • Deletions remove only the 5' region of the HBB promoter, which also results in high levels of HbA2.

• Co-transmission of hereditary persistence of fetal hemoglobin (HPFH), which is the result of single point mutations of the hemoglobin Gγ (HBG2) or hemoglobin Aγ (HBG1) gene promoter. The most common is a single-base substitution C to T at position 158 upstream of the transcription start site of the HBG2 gene, which is silent in normal individuals and in β-thalassemia heterozygotes, but leads to increased HbF production in individuals with erythropoietic stress, as occurs in homozygousβ-thalassemia. This HBG2 mutation, sometimes referred to as Gγ-158 C>T, is officially designated c.-210C>T (reference sequence NM_00184.2). It is in linkage disequilibrium (in cis configuration) with the HBB mutations (see Table 4 and Table 5). This explains the mild phenotype that may result from the inheritance of these mutations.

• Coinheritance of heterocellular HPFH may or may not be linked to the HBB gene cluster. To date, three loci have been mapped: one at Xq22.2-q22.3, one on 6q22.3-q23.1, and one on 8q, which interacts with the HBG2 c.-210C>T mutation). It is likely that many others also exist.

Other modifying factors. The clinical phenotype of homozygous β-thalassemia may also be modified by the coinheritance of other genetic factors mapping outside the β-globin gene cluster. The best known of these modifying genes is the mutation causing Gilbert disease [i.e., (TA)₇ configuration instead of the (TA)₆ in the TATA box of the gene encoding uridin-diphosphoglucuronyltransferase, which, when combined with thalassemia major or thalassemia intermedia, leads to increased jaundice and increased risk of gallstones [Galanello et al 2001]. Less defined modifying factors are genes coding for HFE-associated hereditary hemochromatosis and genes involved in bone metabolism.

In some instances, heterozygous β-thalassemia may lead to the phenotype of thalassemia intermedia instead of the asymptomatic carrier state. Known molecular mechanisms include the following:

• Heterozygosity for mutations in HBB that result in hyper-unstable hemoglobins (dominant β-thalassemia), which precipitate in the red cell membrane together with unassembled hemoglobin alpha chains, resulting in markedly ineffective erythropoiesis. Most of these HBB mutations lie in the third exon and lead to the production of a markedly unstable Hb variant often not detectable in peripheral blood.

• Compound heterozygosity for typical β-thalassemia mutations and the triple or (less frequently) quadruple alpha gene arrangement (αα/αα or ααα/ααα or αααα/αα may increase the imbalance in the ratio of globin alpha/non-globin alpha chains.

Prevalence

β-thalassemia is prevalent in populations in the Mediterranean, Middle East, Transcaucasus, Central Asia, Indian subcontinent, and Far East. It is also common in populations of African heritage. The highest incidences are reported in Cyprus (14%), Sardinia (12%), and southeast Asia.

The high gene frequency of β-thalassemias in these regions is most likely related to the selective pressure from malaria [Flint et al 1998]. This distribution is quite similar to that of endemic Plasmodium falciparum malaria. However, because of population migration and, in a limited part, the slave trade, β-thalassemia is now also common in northern Europe, North and South America, the Caribbean, and Australia.

Evaluations Following Initial Diagnosis

The initial step following diagnosis of β-thalassemia is to distinguish thalassemia intermedia from thalassemia major (see Testing Strategy). The diagnosis of thalassemia major implies the need for a regular transfusion program; the diagnosis of thalassemia intermedia implies the need for intermittent transfusions on an as-needed basis.

Treatment of Manifestations

A comprehensive review of the management of thalassemia major and thalassemia intermedia has been published by Thalassemia International Federation and is available at the TIF Web site [Eleftheriou 2000].

Thalassemia major. Regular transfusions correct the anemia, suppress erythropoiesis, and inhibit increased gastrointestinal absorption of iron. Before starting the transfusions, it is absolutely necessary to carry out hepatitis B vaccination and perform extensive red blood cell antigen typing, including Rh, Kell, Kidd, and Duffy and serum immunoglobulin determination, the latter of which detects individuals with IgA deficiency who need special (repeatedly washed) blood unit preparation before each transfusion. The transfusion regimen is designed to obtain a pre-transfusion Hb concentration of 95-100 g/L. Transfusions are usually given every two to three weeks.

Thalassemia intermedia. Treatment of individuals with thalassemia intermedia is symptomatic and based on splenectomy and folic acid supplementation. Treatment of extramedullary erythropoietic masses is based on radiotherapy, transfusions, or, in selected cases, hydroxyurea (with a protocol similar to that used for sickle cell disease). Hydroxyurea also increases globin gamma chains and may have other undefined mechanisms. Because individuals with thalassemia intermedia may develop iron overload from increased gastrointestinal absorption of iron or from occasional transfusions, chelation therapy is started when the serum ferritin concentration exceeds 300 µg/L.

Bone marrow transplantation

• Bone marrow transplantation (BMT) from an HLA-identical sib represents an alternative to traditional transfusion and chelation therapy. If BMT is successful, iron overload may be reduced by repeated phlebotomy, thus eliminating the need for iron chelation.

• The outcome of BMT is related to the pretransplantation clinical conditions, specifically the presence of hepatomegaly, extent of liver fibrosis, and magnitude of iron accumulation. In children who lack the above risk factors, disease-free survival is over 90% [Gaziev & Lucarelli 2003]. A lower survival rate of approximately 60% is reported in individuals with all three risk factors.

• Chronic graft-versus-host disease (GVHD) of variable severity may occur in 5%-8% of individuals.

• BMT from unrelated donors has been carried out on a limited number of individuals with β-thalassemia. Provided that selection of the donor is based on stringent criteria of HLA compatibility and that individuals have limited iron overload, results are comparable to those obtained when the donor is a compatible sib [La Nasa et al 2005]. However, because of the limited number of individuals enrolled, further studies are needed to confirm these preliminary findings.

Cord blood transplantation. Cord blood transplantation from a related donor offers a good probability of a successful cure and is associated with a low risk of GVHD [Locatelli et al 2003, Walters et al 2005]. For couples who have already had a child with thalassemia and who undertake prenatal diagnosis in a subsequent pregnancy, prenatal identification of HLA compatibility between the affected child and an unaffected fetus allows collection of placental blood at delivery and the option of cord blood transplantation to cure the affected child [Orofino et al 2003]. On the other hand, in case of an affected fetus and a previous normal child, the couple may decide to continue the pregnancy and pursue BMT later, using the normal child as the donor.

Prevention of Primary Manifestations

Early detection of anemia, the primary manifestation of the disease, allows early appropriate treatment and monitoring.

Prevention of Secondary Complications

Transfusional iron overload. The most common secondary complications are those related to transfusional iron overload, which can be prevented by adequate iron chelation. After ten to 12 transfusions, chelation therapy is initiated with desferrioxamine B (DFO) administered five to seven days a week by 12-hour continuous subcutaneous infusion via a portable pump. Recommended dosage depends on the individual's age and the serum ferritin concentration. Young children start with 20-30 mg/kg/day, increasing up to 40 mg/kg/day after age five to six years. The maximum dose is 50 mg/kg/day after growth is completed. The dose may be reduced if serum ferritin concentration is low. By maintaining the total body iron stores below critical values (i.e., hepatic iron concentration <7.0 mg per gram of dry weight liver tissue), desferrioxamine B therapy prevents the secondary effects of iron overload, resulting in a consistent decrease in morbidity and mortality [Borgna-Pignatti et al 2004].

Ascorbate repletion (daily dose not to exceed 100-150 mg) increases the amount of iron removed after DFO administration.

Side effects of DFO chelation therapy are more common in the presence of relatively low iron burden and include ocular and auditory toxicity, growth retardation, and, rarely, renal impairment and interstitial pneumonitis. DFO administration also increases susceptibility to Yersinia infections. The major drawback of DFO chelation therapy is low compliance resulting from complications of administration.

In clinical practice, the effectiveness of DFO chelation therapy is monitored by routine determination of serum ferritin concentration. However, serum ferritin concentration is not always reliable for evaluating iron burden because it is influenced by other factors, the most important being the extent of liver damage.

Determination of liver iron concentration in a liver biopsy specimen shows a high correlation with total body iron accumulation and is the gold standard for evaluation of iron overload. However, (1) liver biopsy is an invasive technique involving the possibility (though low) of complications; (2) liver iron content can be affected by hepatic fibrosis, which commonly occurs in individuals with iron overload and HCV infection; and (3) irregular iron distribution in the liver can lead to possible false-negative results [Clark et al 2003].

In recent years, MRI techniques for assessing iron loading in the liver and heart have improved [Anderson et al 2001, Wood et al 2004, St Pierre et al 2005]. T2 and T2* parameters have been validated for liver iron concentration. Cardiac T2* is reproducible, is applicable between different scanners, correlates with cardiac function, and relates to tissue iron concentration [Anderson et al 2001, Wood et al 2004]. Clinical utility of T2* in monitoring individuals with siderotic cardiomyopathy has been demonstrated [Anderson et al 2004]. Calibration of T2* in the heart will be available in the near future.

Magnetic biosusceptometry (SQUID), which gives a reliable measurement of hepatic iron concentration, is another option [Fischer et al 2003]; however, magnetic susceptometry is presently available only in a limited number of centers worldwide.

Two other chelators have been introduced into clinical use: deferiprone and desferrioxamine.

• Deferiprone (L-1), a bidentated oral chelator, available for several years in many countries, is administered in a dose of 75-100 mg/kg/day. The main side effects of deferiprone therapy include neutropenia, agranulocytosis, arthropathy, and gastrointestinal symptoms [Cohen et al 2003] that demand close monitoring. Recent findings seem to exclude any correlation between deferiprone treatment and progression of liver fibrosis [Wanless et al 2002]. The effect of deferiprone on liver iron concentration may vary among the individuals treated. However, results from independent studies suggest that deferiprone may be more cardioprotective than desferrioxamine; compared to those being treated with DFO, individuals being treated with deferiprone have better myocardial MRI pattern and less probability of developing (or worsening pre-existing) cardiac disease [Anderson et al 2002, Piga et al 2003]. These retrospective observations have been confirmed in a prospective study [Pennell et al 2006].

  After many years of controversy, deferiprone is emerging as a useful iron chelator equivalent/alternative to desferrioxamine.

• Deferasirox recently became available for clinical use in patients with thalassemia. It is effective in adults and children and has a defined safety profile that is clinically manageable with appropriate monitoring. The most common treatment-related adverse events are gastrointestinal disorders, skin rash, and a mild, non-progressive increase in serum creatinine concentration. Post-marketing experience and several phase IV studies will further evaluate the safety and efficacy of deferasirox.

New strategies of chelation using a combination of desferrioxamine and deferiprone have been effective in individuals with severe iron overload; toxicity was manageable [Wonke et al 1998, Wu et al 2004, Tanner et al 2007].

Cardiac disease. In the past few years, particular attention has been directed to the early diagnosis and treatment of cardiac disease because of its critical role in determining the prognosis of individuals with β-thalassemia. Assessment of myocardial siderosis and monitoring of cardiac function combined with intensification of iron chelation can result in excellent long-term prognoses [Anderson et al 2004, Davis et al 2004].

Osteoporosis. Osteoporosis is a common complication in adults with thalassemia major and intermedia [Voskaridou & Terpos 2004]. Its origin is multifactorial, making it difficult to manage. Treatment involves appropriate hormonal replacement, an effective regimen of transfusion and iron chelation, calcium and vitamin D administration, and regular physical activity. Specific treatment with bisphosphonates has been attempted with promising results in several studies and confirmed in a randomized trial [Voskaridou et al 2006].

Surveillance

For individuals with thalassemia major, follow-up to monitor the effectiveness of transfusion therapy and chelation therapy and their side effects includes the following:

• Physical examination every month by a physician familiar with the affected individual and the disease

• Assessment of liver function tests (serum concentration of ALT) every two months

• Determination of serum ferritin concentration every three months

• Assessment of growth and development every six months

• Annual

  • Ophthalmologic and audiologic examinations

  • Complete cardiac evaluation, and evaluation of thyroid, endocrine pancreas, parathyroid, adrenal, and pituitary function

  • Liver ultrasound evaluation and determination of serum alpha-fetoprotein concentration in adults with hepatitis C and iron overload for early detection of hepatocarcinoma

  • Bone densitometry to assess for osteoporosis in the adult

• Regular gallbladder echography for early detection of cholelithiasis [Galanello et al 2001], particularly in individuals with the Gilbert syndrome genotype [i.e., presence of the (TA)7/(TA)7 motif in the promoter of the UGT1A gene]

Agents/Circumstances to Avoid

The following should be avoided:

• Alcohol consumption, which in individuals with liver disease has a synergistic effect with iron-induced liver damage

• Iron-containing preparations

Testing of Relatives at Risk

If prenatal diagnosis has not been utilized and if the disease-causing mutations have been identified in an affected family member, it is appropriate to offer molecular genetic testing to at-risk sibs to allow early diagnosis and appropriate treatment.

See Genetic Counseling for issues related to testing of at-risk relatives for genetic counseling purposes.

Therapies Under Investigation

New chelation strategies, including the combination or alternate treatment with the available chelators, are under investigation.

Induction of fetal hemoglobin synthesis can reduce the severity of β-thalassemia by improving the imbalance between alpha and non-alpha globin chains. Several pharmacologic compounds including 5-azacytidine, decytabine, and butyrate derivatives have had encouraging results in clinical trials [Pace & Zein 2006]. These agents induce Hb F by different mechanisms that are not yet well defined. Their potential in the management of β-thalassemia syndromes is under investigation.

The efficacy of hydroxyurea treatment in individuals with thalassemia is still unclear. Hydroxyurea is used in persons with thalassemia intermedia to reduce extramedullary masses, to increase hemoglobin levels, and, in some cases, to improve leg ulcers. A good response, correlated with particular polymorphisms in the beta-globin cluster (i.e., C>T at -158 G gamma), has been reported in individuals with transfusion dependence [Bradai et al 2003, Yavarian et al 2004]. However, controlled and randomized studies are warranted to establish the role of hydroxyurea in the management of thalassemia syndromes.

The possibility of correction of the molecular defect in hematopoietic stem cells by transfer of a normal gene via a suitable vector or by homologous recombination is being actively investigated [Sorrentino & Niehuis 2001]. The most promising results in the mouse model have been obtained with lentiviral vectors [Rivella et al 2003, Puthenveetil et al 2004].

Search ClinicalTrials.gov for access to information on clinical studies for a wide range of diseases and conditions.

Other

Attempts at in utero transplantation using the father as a haploidentical donor have consistently failed.

Genetics clinics are a source of information for individuals and families regarding the natural history, treatment, mode of inheritance, and genetic risks to other family members as well as information about available consumer-oriented resources. See the GeneTests Clinic Directory.

Support groups have been established for individuals and families to provide information, support, and contact with other affected individuals. The Resources section may include disease-specific and/or umbrella support organizations.

Mode of Inheritance

The β-thalassemias are inherited in an autosomal recessive manner.

Risk to Family Members

Parents of a proband

• The parents of an affected child are obligate heterozygotes and, therefore, carry a single copy of a disease-causing HBB mutation.

• Heterozygotes are clinically asymptomatic but occasionally slightly anemic. Carriers are often referred to as having thalassemia minor (orβ-thalassemia minor).

Sibs of a proband

• At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier.

• Once an at-risk sib is known to be unaffected, the chance of his/her being a carrier is 2/3.

• Heterozygotes are clinically asymptomatic but occasionally slightly anemic. Carriers are often referred to as having thalassemia minor (orβ-thalassemia minor).

Offspring of a proband

• Each child of a proband inherits one copy of a disease-causing HBB mutation from the affected parent and thus is an obligate heterozygote.

• Because of the high carrier rate for β-thalassemia in certain populations (see Prevalence), the offspring of an affected individual and a reproductive partner from one of the high-prevalence areas are at increased risk forβ-thalassemia.

• Given the high carrier rate for β-thalassemia in these populations, it is appropriate to offer carrier testing to the partner of a proband with β-thalassemia.

Other family members of a proband. Each sib of the proband's parents is at a 50% risk of being a carrier.

Carrier Detection

Individuals who should be considered for carrier detection:

• Family members

• Gamete donors

• Members of at-risk ethnic groups (see Table 4) [Cao et al 1997]

Hematologic testing. The carrier state is often referred to as β-thalassemia minor. Carriers are often identified by analysis of red blood cell indices (Table 1), which shows microcytosis (low MCV) and reduced content of Hb per red cell (low MCH), and by quantitative Hb analysis (Table 2), which displays HbA₂ greater than 3.5%.

Pitfalls in carrier identification by hematologic testing:

• Coinheritance of α-thalassemia, which may normalize the red blood cell indices. However, in α/β double heterozygotes, the HbA₂ concentration remains in the β-thalassemia carrier range and thus is of diagnostic value.

• Coinheritance of δ-thalassemia, which reduces to normal the increased Hb A₂ levels typical of the β-thalassemia carrier state. Double heterozygosity for δ- and β-thalassemia can be distinguished from the most common α-thalassemia carrier state by globin chain synthesis or globin gene analysis.

• Confusion of α-thalassemia carriers with β-thalassemia carriers, resulting from microcytosis and hypochromia. However, α-thalassemia carriers are easily distinguished by normal HbA₂ levels (see Alpha-Thalassemia).

• Silent HBB mutations, i.e., very mild mutations associated with consistent residual output of hemoglobin beta chains and with normal RBC indices and normal or borderline HbA₂. However, homozygosity for silent mutations or compound heterozygosity for a silent HBB mutation and a typical HBB mutation result in mild non-transfusion-dependent forms of β-thalassemia.

Molecular genetic testing. When the hematologic analysis is abnormal, molecular genetic testing of HBB is performed to identify the disease-causing mutation. Mild and silent β-thalassemia mutations, which may result in attenuated forms of the disease, are identified and thus may lead to improved genetic counseling of couples at risk.

Population Screening

Individuals at increased risk. Because of the high carrier rate for HBB mutations in certain populations and the availability of genetic counseling and prenatal diagnosis, population screening is ongoing in several at-risk populations in the Mediterranean [Cao et al 2002]. Carrier testing relies on hematologic analysis. When the hematologic analysis indicates a β-thalassemia carrier state, molecular genetic testing of HBB can be performed to identify a disease-causing mutation. If both partners of a couple have the HBB disease-causing mutation, each of their offspring has a 1/4 risk of being affected. Through genetic counseling and the option of prenatal testing, such a couple can opt to bring to term only those pregnancies in which the fetus is unaffected.

Related Genetic Counseling Issues

See Management, Testing of Relatives at Risk for information on testing at-risk relatives for the purpose of early diagnosis and treatment.

Family planning. The optimal time for determination of genetic risk, clarification of carrier status, and discussion of the availability of prenatal testing is before pregnancy. It is appropriate to offer genetic counseling (including discussion of potential risks to offspring and reproductive options) to young adults who are affected or are carriers.

DNA banking. DNA banking is the storage of DNA (usually extracted from white blood cells) for possible future use. Because it is likely that testing methodology and our understanding of genes, mutations, and diseases will improve in the future, consideration should be given to banking DNA of affected individuals. DNA banking is particularly relevant in situations in which the reasons of a particular phenotype are not defined. See for a list of laboratories offering DNA banking.

Prenatal Testing

High-risk pregnancies

• Molecular genetic testing. Prenatal diagnosis for pregnancies at increased risk is possible by analysis of DNA extracted from fetal cells obtained by amniocentesis usually performed at approimately 15-18 weeks' gestation or chorionic villus sampling (CVS) at approximately ten to 12 weeks' gestation. Both disease-causing alleles must be identified before prenatal testing can be performed.

• Preimplantation genetic diagnosis. Preimplantation genetic diagnosis (PGD) may be available for families in which the disease-causing mutations have been identified. For laboratories offering PGD, see .

• Analysis of fetal cells in maternal blood. Prenatal diagnosis by analysis of fetal cells in maternal blood is not yet available, but is being investigated on a research basis [Mavrou et al 2007].

• Analysis of fetal DNA in maternal plasma for the presence of the father's mutation may lead to prenatal exclusion of homozygous β-thalassemia. This testing is not yet clinically available, but is being investigated on a research basis with promising results [Lo 2005].

Indeterminate-risk pregnancies. An indeterminate risk pregnancy is one in which:

• One parent is a definite heterozygote and the other parent has a β-thalassemia-like hematologic picture, but no HBB mutation has been identified by sequence analysis.

• A mother is a known heterozygote and the father is unknown or unavailable for testing, especially if the father belongs to a population at risk.

In either instance, the options for prenatal testing should be discussed in the context of formal genetic counseling. In indeterminate-risk pregnancies, the prenatal testing strategy is analysis of DNA extracted from fetal cells obtained by amniocentesis usually performed at approximately 15-18 weeks' gestation or chorionic villus sampling (CVS) at approximately ten to 12 weeks' gestation for the known HBB mutation. If the known HBB mutation is present, analysis of globin chain synthesis is performed on a fetal blood sample obtained by percutaneous umbilical blood sampling (PUBS) at approximately 18-21 weeks' gestation.

Note: Gestational age is expressed as menstrual weeks calculated either from the first day of the last normal menstrual period or by ultrasound measurements.

Literature Cited

Published Statements and Policies Regarding Genetic Testing

No specific guidelines regarding genetic testing for this disorder have been developed.

Suggested Readings

Revision History

• 23 October 2007 (me) Comprehensive update posted to live Web site

• 19 June 2005 (me) Comprehensive update posted to live Web site

• 4 April 2003 (ac) Revision

• 18 March 2003 (tk) Comprehensive update posted to live Web site

• 28 September 2000 (me) Review posted to live Web site

• March 2000 (ac) Original submission